Case report HIGH GRADE LYMPHOMA IN A SIX-YEAR OLD …tru.uni-sz.bg/bjvm/B. Emikpe OnFirst.pdf · 2016-08-05 · Morphologically, the involved lymph nodes showed a diffuse effacement

Bulgarian Journal of Veterinary Medicine, 2016 ONLINE FIRST ISSN 1311-1477; DOI: 10.15547/bjvm.1021

Case report

HIGH GRADE LYMPHOMA IN A SIX-YEAR OLD BOERBOEL: A CASE REPORT

B. O. EMIKPE1, T. O. OMOBOWALE2, T. A. JARIKRE1, P. I. OTUH3, V. O. OYEBANJI1 & R. E. ANTIA1

1Department of Veterinary Pathology, Faculty of Veterinary Medicine, University of Ibadan, Nigeria; 2Department of Veterinary Medicine,

Faculty of Veterinary Medicine, University of Ibadan, Nigeria; 3Small Animal Clinic, Veterinary Teaching Hospital, University of Ibadan, Nigeria

Summary

Emikpe, B. O., T. O. Omobowale, T. A. Jarikre, P. I. Otuh, V. O. Oyebanji & R. E. Antia, 2016. High grade lymphoma in a six-year old Boerboel: A case report. Bulg. J. Vet. Med. (online first). A case of high grade large cell lymphoma was diagnosed in a dog at the Veterinary Teaching Hospi-tal, University of Ibadan. The animal was a male Boerboel over six years old. The case was monitored clinically until necropsy where cytological, gross and histological techniques were used for detailed examination of morphological features of the tumour. The morphological pattern and classification schemes of lymphoma were generally reviewed. The relevance of other diagnostic methods was em-phasised.

Key words: canine, diagnosis, lymphoma, pathology

Lymphoma is one of the haematopoietic malignancies encountered in small ani-mals. It accounts for 7% to 24% of all canine tumours and up to 83% of all ca-nine haematopoietic malignancies (Vail, 2010; Zandvliet, 2016). However, apart from reports of splenic lymphoma in a local bitch (Oni et al., 2004) and another CD33 positive lymphoma in an Alsatian, very little is known on the epidemiology of canine lymphoma in Nigeria.

Clinical signs and physical examina-tion are often suggestive of lymphoma or

lymphosarcoma (LSA). The definitive diagnosis requires cytology, histopatho-logy and/or molecular diagnostics (Couto, 2009).

A number of classification schemes have been developed over the years, how-ever, both the revised European American (REAL) and the very similar WHO sys-tems are appropriate for the classification of animal haematopoietic neoplasms. The morphological presentation of the neo-plastic cells still remains crucial in diag-nosis of lymphoma in animals (Ponce et

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al, 2010). Other factors to consider in-clude history and physical examination, complete blood count and serum chemis-try, urinalysis, and/or radiography. The clinical picture and pathological features observed in this case were described to further understand the subtle characteristic presentation and epidemiology of canine lymphoma in our environment.

The dog, a male Boerboel over six years old with brown fur was presented to the Small Animal Clinic of the Veterinary Teaching Hospital, University of Ibadan with a complaint of inability to bear weight on the right hind limb. The dog had been recumbent since the condition was first noticed. Clinical signs observed included: swelling of the inguinal and anal regions, bilateral muco-purulent ocular discharges, and marked distention of the abdomen. The dog was also unable to urinate (stranguria) due to compression in the inguinal region. There was pedal oe-dema, dyspnoea, weak pulse and faint heart sound on auscultation. The 6 lead ECG report showed cardiac arrhythmia and prolonged Q-T wave (Fig. 1) indica-

tive of cardiac abnormality. The haemato-logical and serum biochemical values were within the normal range for the spe-cies. Fine needle aspiration (FNA) cyto-logy was equally carried out as well as coprology which revealed presence of 2–3 Isospora canis per high power field.

Ultrasonography of the abdominal re-gion revealed a few abdominal masses. The weight of the animal dropped from 75 kg on presentation to 52 kg before death in space of two weeks. A tentative cinical diagnosis of multi-organ failure was made.

On necropsy, the carcass was fresh. There were several ticks on the body of the carcass, it was moderately emaciated. The oral and ocular mucous membranes were pale. There was widespread enlargement of superficial lymph nodes (submandibular, axillary, popliteal and pre-femoral) (Fig. 2A). The abdomen was markedly distended (caudally from the position of the last set of ribs). There were multiple raised dark spotted nodules of <2 cm diameter on the lung lobes. The mediastinal lymph nodes were enlarged

Fig. 1. ECG record showing prolonged QT- interval and ventricular premature complexes.

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and haemorrhagic on cut surface. An ir-regular spherical large nodule (>2.5 cm diameter) was seen on the base of the heart while the left ventricular wall was enlarged and distended with irregular greyish areas (infiltrative nodules). The muscular part of the diaphragm was mark-edly thickened with foci of neoplastic infiltrates and nodules. The abdominal cavity contained about 3.5 L of straw col-oured fluid.

The liver was dark red and markedly enlarged with numerous irregular sized creamy coloured deep seated nodules on the hepatic lobes (Fig. 2B). The right lobe

of the liver was adhered to the diaphragm and loops of the pancreas. Multiple dark coloured nodules (<2 cm) were also pre-sent in the pancreas. A firm irregular and large sized tissue mass of firm consistency (7×6 cm) was found in the retroperitoneal fat attached to the right kidney and in the inguinal region (inguinal lymph node). Another similar tissue mass was also found extensively attached to the liver, duodenum, pancreas and muscular dia-phragm. The mesenteric lymph nodes were enlarged with thin cortex and dis-tended capsule and mottled medulla. The spleen was dark red, moderately enlarged

A B

Fig. 2. A. Lymph node with effacement of medulla (asterisk) and haemorrhagic periphery (arrow).

B. Liver with cream coloured nodules (asterisk) on cut surface.

A B

Fig. 3. Large lymphoid cells (arrows) from lymph node (A) and heart nodule (B) with anisocytosis,

prominent nucleoli and high nuclear-cytoplasmic ratio. Giemsa, scale bar=18 µm.

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with a few raised circumscribed nodules. The gastric mucosa was haemorrhagic with multiple ulcers (0.5×1 cm) in the fundus. The intestinal serosa had raised opaque nodules of various size. The entire length of the intestinal mucosa was hae-morrhagic (suffusive) with ulcers at the jejunal region. The prostate gland had nodules of various size which were haem-orrhagic on cut surface. Similar nodules were also present on the kidneys. The urinary bladder mucosa was thickened and had areas of suffusive haemorrhages with ulcers of variable sizes. The bone marrow from the diaphysis of the femur was dif-fusely cherry red in colour.

Cytological smears obtained by FNA and impressions from nodal or extranodal masses (liver, pancreas, lungs, spleen, and prostrate, intestinal serosa, kidney) were air-dried, fixed, and stained using the May Grunwald Giemsa technique. Microscopic examination of Giemsa stained smears from the liver nodules showed high cellu-larity, marked infiltrates of predominantly large to small discrete round (lymphoid) cells. The cells had high nuclear-cytoplasmic (N:C) ratio, marked anisocy-tosis, thin rim of bluish cytoplasm, and prominent to multiple nucleoli. There

were also a few vacuolated hepatocytes, neutrophils and macrophages. Other nodal masses showed preponderance of large lymphoid cells (lymphoblast) having high N:C, narrow/thin rim of cytoplasm, nu-clear moulding, 3–5 mitotic figures/HPF and also anisocytosis. The lymphoblasts accounted for >50% of all nucleated cells present in a lymph node (Fig. 3A). Similar discrete round cells were present in the smears from the muscles, prostate, lungs, peripheral blood, bone marrow, pancreas and intestinal nodules (Fig. 3B).

The cytological diagnosis was metas-tatic round cell tumour (lymphoma), based on the preponderance of lymphoblasts accounting for more than 50% of all nu-cleated cells present in nodal and extra-nodal masses.

Histological evaluation of biopsy specimens from enlarged nodal and ex-tranodal masses were fixed in 10% neutral buffered formalin at room temperature for 48 h and embedded in paraffin wax. Tis-sue sections 5 µm in thickness were stained with haematoxylin and eosin (HE) and examined using light microscope. Morphologically, the involved lymph nodes showed a diffuse effacement of normal architecture with marked thinning

A B

Fig. 4. A. Lymph node showing loss of architecture with marked expansion of the medullary

region (asterisk). HE, scale bar=180 µm. B. Lymph node showing preponderance of large cells (arrow) in the lymphoid follicle. HE, scale bar=45 µm.

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of the peripheral capsule and focal coloni-sation of the capsule and perinodal struc-ture (Fig. 4A). Marked atrophy and co-agulation necrosis of lymphoid follicles, apoptotic bodies, infilling of the paracor-tex extending into the medullary region with numerous tangible body macro-phages, expansion of the medullary cords and compression of the medullary sinuses were noticed (Fig. 4B). There were also lighter areas at a higher level of cellular proliferation. The neoplastic cells had chromocentres separated by parachro-matin areas in the nucleus. The chromatin was densely stained with prominent nu-cleolus. Similar neoplastic cells were dif-fusely infiltrating the liver causing com-pressional atrophy of hepatocytes (Fig. 5A), heart (Fig. 5B) and diaphragmatic muscles, lungs, spleen, intestine and kid-neys. Morphological diagnosis of lym-phoma (LSA) was made.

Grading was determined by the size of cells (majority of large-sized cells) and by mitotic index (MI). Thus, considering the cytological details and morphological criteria based on the updated Kiel histo-logical and cytological classification, a case of high grade large cell lymphoblas-tic lymphoma was identified.

Lymphoma or lymphosarcoma arises due to the proliferation of malignant lym-phoid cells – usually in lymphoid tissue, such as lymph nodes, liver or spleen, but the tumour may originate in any tissue (Couto, 2009; Vail, 2010). This origin from solid organs distinguishes LSA from lymphoid leukaemia, as the latter arises from bone marrow (Couto, 2009). The etiology of LSA is considered to be multi-factorial. However, several environmental, infectious, immune-mediated and genetic factors are associated with a higher risk of developing LSA in other climes (Mortier et al., 2012).

The most frequently observed clinical signs are non-specific including peripheral lymphadenopathy. All the lymph nodes including mandibular and prescapular ones, were affected in this case. The diffi-culty in micturition was due to compres-sion from the nodal, prostatic masses and ensued polypoid cystitis. Haematologic and serum biochemical changes were within normal range in this case, though usually they are not diagnostic (Couto, 2009; Mizutani et al., 2016). Anaemia and thrombocytopaenia, if present, are usually related to bone marrow infiltration, para-neoplastic immune-mediated destruction,

A B

Fig. 5. Liver (A) and heart (B) showing diffuse infiltration of neoplastic cells (arrows) and distor-

tion of normal architecture. HE, scale bar=45 µm.

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splenic infiltration and/or chronic disease. Regenerative anaemia may also be associ-ated with concomitant blood loss (Couto, 2009; Vail, 2010). The ECG abnormality suggested that the tumour induces car-diomyopathy, weakness and exercise in-tolerance of the dog ante mortem. This nature of metastasis to distant organs and tissues confirmed the high grade of the neoplasm.

The defining strategy of the revised European American (REAL) and the very similar WHO systems is that each entity is a fully characterised scheme based on all information including cell type, tumour architecture, topography, age, gender, and phenotype and in some cases genotype (Valli et al., 2013). Both systems recog-nise and separate lesions that may have similar morphology, but different pheno-types and rates of biological progression (Morton et al., 2007). Furthermore, the REAL system can be applied very largely on the basis of routine histochemistry to define B- and T-cell lymphocyte deriva-tion on paraffin embedded tissues (Jaffe et al., 2008). The application of these sys-tems however, to reviews of lymphomas in animals and their response to therapy provide major impetus to the understand-ing of lymphomas in animals from the standpoint of research on spontaneous lymphoid neoplasms of outbred species, as well as treatment of lymphoid tumours in companion animals, which may be very useful in this environment.

In general, lymphoproliferative dis-eases form a spectrum with lymphoma and lymphoid leukaemia at their extremes, and it is not always possible to determine whether the disease in a particular animal is primarily peripheral (lymphoma), or of bone marrow origin (leukaemia). The ma-jor distinction between both is in the tis-sue area with the largest mass of tumour

cells. It is now understood that, in malig-nancies of the lymphoid system, there are always tumour cells in the circulation whether or not they are recognised in the peripheral blood. However, the operative factors in the dissemination of tumours of the lymphoid system are the presence and type of intercellular cytoplasmic adhesion molecules (ICAMs) on the surface of the tumour cells that mediate adhesion to en-dothelium and the ability to emigrate to new tissue areas (Rezuke et al., 1997; Atizadeh et al., 2000). Malignancy in an ontogenically primitive cell is likely to occur in the bone marrow, thus presenting as leukaemia in a young animal or indi-vidual with the disease. In contrast, clonal autonomy in a mature lymphocyte is likely to occur in the peripheral tissues in a ma-ture animal or individual with presentation as a lymphoma.

Moreover, leukaemias present some degree of marrow failure characterised by anaemia, thrombocytopaenia, or neutro-paenia, which occur when 50% or more of the bone marrow is involved by the tu-mour. Under these circumstances, the blood and bone marrow are always diag-nostic. In contrast, the lymphomas which involve peripheral tissues tend to leave the bone marrow relatively uninvolved and, at the time of diagnosis, these animals usu-ally have normal haemoglobin, platelet and leukocyte counts, as observed in this case.

Lymphoma of small lymphocytic cell type (SLL) needs to be differentiated from peripheralising lymphoma of larger lym-phocytes and chronic lymphocytic leu-kaemia which are distinguished by their cytoplasmic granules (Erdman et al., 1995). Another major differential diagno-sis is prolymphocytic leukaemia (PLL). SLL must have less than 10% prolympho-cytes whereas disorders with 10 to 50%

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prolymphocytes are considered chronic lymphocytic leukaemia (CLL) and PLL when greater than 50% of prolymphocytes in the peripheral blood (Zwiebel & Cheson, 1998).

Once a definitive diagnosis is achieved, clinical staging can be done to allow more accurate prognosis. Immuno-phenotyping of canine lymphoma can classify tumours as T-cell (CD3) or B-cell (CD79); the latter being more common and the former associated with a poorer prognosis. The monoclonality of a popula-tion of neoplastic lymphoid cells support a diagnosis of lymphoma. Both B- and T-cells have cognate receptors which enable them to take part in the immune response. PCR amplification of either the T-cell receptor (TCR) or immunoglobulin chains on B-cells will demonstrate either a mixed population (i.e. with a reactive lymphade-nopathy) or a clonal population of cells (Vernau & Moore, 1999). Other diagnos-tic and prognostic markers have been sug-gested including serum levels of alpha 1-acid glycoprotein (AGP) and matrix metalloproteinases 2 and 9 as indicators of relapse. However, new markers and so-phisticated molecular techniques (such as micro and tissue arrays) are developed to provide information for the clinician in the management of canine lymphomas. Hopefully these would be adopted even in poor resource setting like ours to further characterise the epidemiology of this neo-plasm in dogs.

ACKNOWLEDGEMENTS

We wish to thank the technicians both at the Small Animal Clinic and diagnostic units of the Veterinary Teaching Hospital, University of Ibadan for their support during work-up of this case.

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Paper received 23.03.2016; accepted for publication 10.06.2016

Correspondence: Benjamin O. Emikpe Department of Veterinary Pathology University of Ibadan, Nigeria Tel: +234 8066486080 e-mail: [email protected]